Glutamate is widely acknowledged as the most prominent excitatory neurotransmitter used by the mammalian central nervous system. This project employs the hippocampal formation of the rat brain as a test system with which to investigate the mechanisms of glutamate transmission. The present proposal focuses on three presynaptic mechanisms: autoreceptor regulation of the release process, alterations of the release process due to eicosanoid production and the involvement of proline. Recent work suggests that glutamate/aspartate release can be enhanced by activation of the NMDA receptor and depressed by activation of either the ACPD or the AP4 metabotropic receptor. The mechanisms by which these forms of autoregulation operate are not understood. Slice and synaptosomal preparations of hippocampal area CA1 will be used to address this problem. These preparations release both glutamate and aspartate in a Ca2+-dependent manner from terminals of the extrinsic Schaffer collateral-commissural-ipsilateral associational pathway. Release of these excitatory amino acids will be quantitated during depolarization of CA1 synaptosomes with agents that work by different mechanisms (elevated K+, 4-aminopyridine, veratridine). In parallel experiments, terminal depolarization, [Ca2+]i and synaptic vesicle cycling will be measured. By studying the effects of receptor ligands on these measures, it will be possible to determine some of the cellular processes involved in autoregulation. Eicosanoids (arachidonic acid and its metabolites) are produced and released in large amounts during neuropathological states, and they may mediate the NMDA recep- tor-dependent enhancement of release. Eicosanoids released from the CA1 area during NMDA receptor activation will be identified, their possible involvement in NMDA receptor-mediated autoregulation will be assessed and their effects on the release process will be determined. Finally, a method will be developed to evoke and quantitate the release of glutamate and aspartate from area CA1 in vitro by stimulation of excitatory afferent fibers. This method will be used to determine the physiological and/or pathological conditions under which autoreceptor regulation of glutamate/aspartate release can be detected. Proline is an excitatory and excitotoxic imino acid that can block memory formation. Na+-dependent, high affinity uptake of proline is expressed by a subset of glutamate pathways. These properties argue that proline plays a role in excitatory transmission. Studies of glutamate synthesis and release and of excitatory synaptic transmission will be carried out to elucidate this role. An important tool will be antibodies directed against the presumed transport site of the cloned transporter. Findings from these studies will be relevant to the treatment of neurological conditions, such as epilepsy, which involve hyperactivity of the hippocampal formation, as well as to the etiology of childhood seizures associated with hyperprolinemia. They may also prove relevant to synaptic mechanisms of memory and learning.